Identification of immune related genes in Atlantic halibut (Hippoglossus hippoglossus L.) following in vivo antigenic and in vitro mitogenic stimulation

To identify and characterize genes and proteins of the Atlantic halibut (Hippoglossus hippoglossus) immune system, six cDNA libraries were constructed from liver, kidney, spleen, peripheral blood, and thymus. Halibut were injected with nodavirus, infectious pancreatic necrosis virus (IPNV), or vibriosis vaccine and tissue samples were collected at various time points. Leukocytes from peripheral blood and spleen from stimulated and mock-injected fish were isolated and further in vitro activated with the mitogens, concanavalin A (Con A) and phorbol myristate acetate (PMA) to facilitate activation and proliferation. A total of 5117 high quality expressed sequence tags (ESTs) were identified and assembled into 781 contigs and 2796 singletons. Amongst these ESTs, 147 different putative immune related genes were identified. Several genes involved in innate and adaptive immune responses such as complement proteins, immunoglobulins, cell surface receptors, and cytokines and chemokines were identified. Of the immune related genes identified in this study, 44% had no match against any of the publicly available sequence data for halibut and thus can be considered as novel identification in halibut species. The approach of combining in vivo antigenic with in vitro mitogen stimulation, in addition to preparation of cDNA libraries from thymus enabled identification of many of the interesting genes including those involved in T-cell receptor complex.